Antagonistic interaction between miR-143 and KRAS gene regulating male mouse germ cell apoptosis

Abstract

Precisely regulated spermatocyte growth, differentiation, and apoptosis are crucial for sustainable male fertility. miR-143 has been demonstrated to regulate gene expression and cell apoptosis in various human cancers. However, the function of mmu-mir-143 (miR-143) in mammalian testes and its underlying mechanism remains unexplored. In this study, the expression of miR-143 was detected in C57BL/6 mice spermatocytes by in situ hybridization (ISH) and immunofluorescence (IF) co-staining and transfecting miR-143 inhibitor into GC-2 cells (mouse spermatogenic cells) shows that miR-143 inhibits cleaved Caspase 3 (CC3)-induced male germ cell death. The current study used IF co-staining of KI67 and γ-H2A.X in the testes of C57BL/6 mice at different developmental stages, revealing that active proliferation and apoptosis of spermatocytes occurred simultaneously in the testes at 14 day post-partum (dpp). Kras was predicted as a potential target of miR-143 in mice using of the online database TargetScan, verified by quantitative real-time PCR (qPCR), western blotting (WB), and Dual-luciferase reporter gene assay. Co-transfection of miR-143 inhibitor and Kras siRNA into GC-2 cells revealed an antagonistic correlation between miR-143 and Kras in regulating male germ cell death. Finally, miR-143 inhibitor and mimics were administered into the seminiferous tubule of 3-week-old C57BL/6 mice. The histomorphology, IF co-staining, and WB data indicated that the testes treated with the miR-143 inhibitor showed significantly aberrant phenotypes, including damaged seminiferous tubules, reduced spermatocyte quantity, and elevated levels of apoptosis. This study uncovered the mechanism by which miR-143 inhibits male germ cell apoptosis through the repression of Kras/KRAS levels and the inhibition of Caspase 3 activation, providing insight into the role of miRNA in spermatogenesis and the maintenance of male fertility.